The molecular cell biology of human- and hominoid-specific genes is virtually a terra incognita in basic research. Understanding the molecular cell biology of human-specific genes will open a new vista of research of wide import to understanding human biology and disease. TBC1D3 is a widely expressed hominoid-specific gene, and recent work indicates that TBC1D3 is a powerful stimulant of the EGF and IGF-1 receptor pathways. Thus, expression of TBC1D3 promotes cellular proliferation and enhances the activation of Ras in response to growth factors. TBC1D3 contains a TBC domain that interacts with the small GTPase Rab5, a key regulator of endocytosis. We speculate that TBC1D3 may be both a growth factor receptor regulator and a Rab5 effector. This proposal focuses upon defining the biochemical mechanisms that are set in motion by TBC1D3 and result in cell proliferation. In Specific Aim 1, we will carry out a structure-function study to determine domains in TBC1D3 involved in cell localization and in Ras activation. To understand the linkage between TBC1D3 and the Ras pathway, we will identify serine/threonine and tyrosine-phosphorylation sites on TBC1D3 and validate the role TBC1D3-interacting proteins, Grb2 and 14-3-3, in Ras activation. In Specific Aim 2, we will quantify the interaction between TBC1D3 and the Rab5 subfamily of GTPases. By modeling the TBC domain of TBC1D3 using 3-dimensional structures of known TBC domains, we will identify key residues at the TBC1D3:Rab5 interface and test their role in Rab5 binding. To confirm and extend these initial structural studies, the structure of the TBC domain will be solved. In Specific Aim 3, we hypothesize that TBC1D3 stimulates cell signaling/proliferation by modulating IRS1 phosphorylation and via the interaction between TBC1D3 and the E3 ubiquitin ligase Cul7. We will characterize the effect of TBC1D3 on IRS1 phosphorylation and turnover. To understand the role of Cul7 in TBC1D3 function, we will determine the domains that mediate TBC1D3-Cul7 interaction and using deletion mutants, determine the function of the TBC1D3-Cul7 complex. The cell and molecular biology of TBC1D3 may represent a model to understand the evolution of human-specific genes that function to modulate and regulate evolutionarily conserved cell signaling pathways. PUBLIC HEALTH RELEVANCE: TBC1D3 is a hominoid-specific gene that regulates growth factor receptor signaling. TBC1D3 has been shown to be an oncogene and to be over-expressed in certain proliferative disorders. Human specific genes have not been extensively studied in spite of their relevance to the human condition and their potential role in human specific diseases. This work proposes to elucidate the mechanism of action of TBC1D3 at the cellular and molecular level. TBC1D3 may serve as a model for developing an understanding of the function of human-specific genes in regulating complex signaling pathways.